The mechanisms used to establish and maintain silent chromatin structures are of considerable interest because of their direct relevance for our understanding of diverse processes including development, oncogenesis, and epigenetic reprogramming. Ikaros and the Ikaros family members Aiolos and Helios are highly homologous, sequence-specific DNA-binding proteins that localize to foci of pericentromeric heterochromatin in the interphase nuclei of hematopoietic cells. Several cellular genes are physically repositioned to these foci when they are silenced during lymphocyte development. These results have led to two hypotheses: 1) the pericentromeric repositioning of silent genes may be an essential step in the assembly of silent chromatin, and 2) Ikaros may play an important role in gene silencing and/or pericentromeric repositioning. One speculative model is that Ikaros may bind recognition sites in the control regions of genes destined for silencing and help recruit these genes to pericentromeric foci through the formation of multimeric structures with pericentromeric Ikaros. Rigorous tests of these hypotheses have been difficult to design because of our limited fundamental knowledge of gene silencing during development, because of limited knowledge of the properties of Ikaros, because lymphocytes lacking functions provided by Ikaros are often non-viable, and because methods to selectively disrupt pericentromeric repositioning are not available. We plan to continue our efforts to overcome these limitations. The proposed studies should ultimately lead to rigorous tests of the above hypotheses, and also provide much broader insight into molecular mechanisms of gene silencing during development. The proposed studies will benefit from our recent success in establishing the ex vivo maturation of double-positive (DP) thymocytes as a model for studying the temporal assembly of silent chromatin during developmental, by using the terminal transferase (Dntt) gene as an initial example of a gene that is silenced during this step. To accomplish our objectives, we will first characterize in greater depth the mechanism by which the Dntt locus becomes assembled into silent chromatin during thymocyte maturation. This analysis will address a number of critical, yet straightforward questions, but will also include attempts to develop in vitro silencing and somatic cell genetic assays. In the second aim, the Ptcra gene will be used as an alternative model for studying gene silencing during thymocyte maturation, to overcome specific limitations of the Dntt gene. In the final aim, our characterization of the fundamental properties of Ikaros proteins will be continued, to gain further insight into the functions of this intriguing family of DNA-binding proteins and their potential roles in gene silencing and pericentromeric repositioning.